Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Abstract

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATPbound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.muscle | actomyosin | light chains | fluorescence resonance energy transfer A central challenge in the biophysics of motility is to understand the molecular mechanisms of structural transitions in the actomyosin ATPase (enzyme code 3.6.4.1) cycle, in which myosin alters its affinity for actin in a nucleotide-dependent manner from weak (W) actin-binding states (with ATP or ADP-P i bound to the myosin active site) to strong (S) actin-binding states (with ADP or no nucleotide, i.e., rigor), resulting in the generation of force. It is particularly important to obtain direct information about the structural dynamics of the whole actomyosin complex, because there is evidence that each protein, when studied separately, undergoes changes in structure and dynamics during transitions from the W to S states (1-5). S states are relatively stable and easy to study, but the W actomyosin complex is less accessible to study, due to its transient and structurally dynamic nature.The myosin heavy chain starts with the N-terminal catalytic domain (CD), which contains the nucleotide-binding pocket and the actin-binding site. A small converter region connects the CD to an extended α-helical segment that forms the core of the lightchain domain (LCD) (6, 7). The LCD contains two IQ motifs that serve as binding sites for one essential light chain (ELC) and one regulatory light chain (RLC) and is proposed to function as a lever arm that amplifies small structural c...

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex

“…For TR-FRET measurements, we used time-correlated single-photon counting (41) or direct waveform recording, as described previously (29,30). Global multiexponential analysis of the TR-FRET data was used to test a series of structural models, as described extensively elsewhere (30,41) This mode of analysis resolves both binding and structural information from TR-FRET data.…”

“…Global multiexponential analysis of the TR-FRET data was used to test a series of structural models, as described extensively elsewhere (30,41) This mode of analysis resolves both binding and structural information from TR-FRET data. SR membranes labeled with D-FKBP were incubated with 200 M S100A1 for 2 h at 22°C before a 30-min incubation at 22°C with 800 nM A-CaM.…”

“…structural states) within a protein complex as well as the population of probes in each structural state (29,30). With A N -CaM, the TR-FRET data corresponding to both RyR isoforms (Fig.…”

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

“…Our previous work showed that when myosin binds to actin, the NTE of fast skeletal or cardiac ELC is in close proximity to actin ( Fig. 1) (4,5). N-terminal amino acids of NTE, specifically the first four amino acids (APKK), are highly conserved among skeletal and cardiac muscles and also across species (6).…”

“…These positively charged lysine residues interact with the negatively charged C terminus glutamates of actin (7). The actin-NTE interaction is functionally relevant, because myosin isoforms having NTEs show higher catalytic efficiency and slower motility on actin (3,8,9).Recent studies from our laboratory have explored the structural basis of ELC-mediated modulation of the actin-myosin interaction (4,5,10). Time-resolved FRET (TR-FRET), using a donor on actin and an acceptor on the A1 NTE of skeletal myosin subfragment 1 (S1), showed that the NTE plays an important role modulating myosin's force-producing powerstroke (4).…”